Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed)
Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed)
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DOI：
10.1038/nbt0102-83
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发表时间：
2002-01-01
影响因子：
46.9
通讯作者：
Glick, BS
中科院分区：
文献类型：
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作者：
Bevis, BJ;Glick, BS
红色荧光蛋白DsRed具有理想的光谱特性，适用于使用绿色荧光蛋白（GFP）的双色实验（1）。但野生型DsRed有几个缺点，包括发色团成熟缓慢和溶解性差（2，3）。为了克服缓慢的成熟，我们使用随机和定向诱变来创建DsRed变体，其成熟速度比野生型蛋白快10-15倍。在位置42处的天冬酰胺至谷氨酰胺的取代大大加速了DsRed的成熟，但也增加了绿色发射的水平。额外的氨基酸取代抑制这种绿色发射，同时进一步加速成熟。为了提高DsRed的溶解度，我们减少了蛋白质N末端附近的净电荷。优化的DsRed变体即使在快速生长的生物体（如酵母）中也能产生明亮的荧光。
The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP)(l). But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility(2,3). To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10-15 times faster than the wild-type protein. An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission. Additional amino acid substitutions suppress this green emission while further accelerating the maturation. To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.